Tetsuji Mori

Inducible gene deletion in astroglia and radial glia—A valuable tool for functional and lineage analysis

Astrocytes are thought to play a variety of key roles in the adult brain, such as their participation in synaptic transmission, in wound healing upon brain injury, and adult neurogenesis. However, to elucidate these functions in vivo has been difficult because of the lack of astrocyte‐specific gene targeting. Here we show that the inducible form of Cre (CreERT2) expressed in the locus of the astrocyte‐specific glutamate transporter (GLAST) allows precisely timed gene deletion in adult astrocytes as well as radial glial cells at earlier developmental stages. Moreover, postnatal and adult neurogenesis can be targeted at different stages with high efficiency as it originates from astroglial cells. Taken together, this mouse line will allow dissecting the molecular pathways regulating the diverse functions of astrocytes as precursors, support cells, repair cells, and cells involved in neuronal information processing.

Slit and glypican-1 mRNAs are coexpressed in the reactive astrocytes of the injured adult brain

The slit family serves as a repellent for growing axons toward correct targets during neural development. A recent report describes slit mRNAs expressed in various brain regions in adult rats. However, their functions in the adult nervous system remain unknown. In the present study, we investigated whether slit mRNAs were expressed in the cryo‐injured brain, using in situ hybridization. All slit family members were expressed at the lesion. Slit2 mRNA was the most intensely expressed in the cells surrounding the necrotic tissue. A double‐labeling study showed that slit2 mRNA was expressed in the glial fibrillary acidic protein (GFAP)‐positive reactive astrocytes. In addition, glypican‐1, a heparan sulfate proteoglycan that serves as a high‐affinity receptor for Slit protein, was coexpressed with slit2 mRNA in the reactive astrocytes. These findings suggested that slit2 might prevent regenerating axons from entering into the lesion in concert with glypican‐1. GLIA 42:130–138, 2003.

The LIM homeobox gene, L3/Lhx8, is necessary for proper development of basal forebrain cholinergic neurons.

Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive functions such as learning and memory, and are affected in several neurodegenerative diseases (e.g. Alzheimers disease). Despite their importance, the molecular mechanisms of their development are not fully elucidated. A recent report demonstrated that some BFCNs in adult rat are positive for L3/Lhx8, a LIM homeobox transcription factor. To examine the function of L3/Lhx8 in the development of BFCNs, we generated L3/Lhx8 gene‐disrupted mice. In these mice, cells expressing cholinergic neuron markers, such as choline acetyltransferase, vesicular acetylcholine transporter and p75 low‐affinity NGF receptor, were markedly reduced in the basal forebrain, whereas other cholinergic neurons including brain stem and spinal motor neurons expressed the markers. Neurotransmitter phenotypes other than cholinergic in the basal forebrain appeared intact. From these results, we suggested that L3/Lhx8 has a pivotal and specific role in the development and/or maintenance of BFCNs.

Increased syndecan expression by pleiotrophin and FGF receptor-expressing astrocytes in injured brain tissue.

Syndecan‐1, ‐2, ‐3, and ‐4 are heparan sulfate proteoglycans that are differentially expressed during development and wound repair. To determine whether syndecans are also involved in brain injury, we examined the expression of syndecan core proteins genes in cryo‐injured mouse brain, using in situ hybridization. All syndecan mRNA transcripts were similarly expressed in the region surrounding the necrotic tissue, exhibiting peak levels at day 7 after injury. Comparison with cellular markers showed that reactive astrocytes were the primary source of syndecans. Syndecans serve as co‐receptors for fibroblast growth factor (FGF) and as a reservoir for another heparin‐binding growth factor, pleiotrophin (PTN, or heparin‐binding growth‐associated molecule. In our model, FGF receptor1 (FGFR1) and PTN mRNA levels were upregulated in reactive astrocytes. The distribution patterns of FGFR1 and PTN overlapped considerably with those of syndecan‐1 and ‐3 mRNAs, respectively. These results suggest that syndecans are expressed primarily in reactive astrocytes, and may provide a supportive environment for regenerating axons in concert with heparin‐binding growth factors (e.g., FGF and PTN) in the injured brain. GLIA 39:1–9, 2002.

Molecular cloning of novel leucine-rich repeat proteins and their expression in the developing mouse nervous system

It is well established that leucine-rich repeat (LRR) proteins such as connectin, slit, chaoptin, and Toll have pivotal roles in neuronal development in Drosophila as cell adhesion molecules. However, to date, little information concerning mammalian LRR proteins has been reported. In the present study, we sought LRR proteins of the mouse brain, based on the assumption that fundamental mechanisms are conserved between different species. We screened a neonatal mouse brain cDNA library with a human partial cDNA encoding LRR protein as a probe. We obtained two independent cDNAs encoding LRR proteins, designated NLRR-1 and NLRR-2 (Neuronal Leucine-Rich Repeat proteins). We analyzed the whole sequence of NLRR-1 and partial sequence of NLRR-2. Sequence analysis showed that these two clones are about 60% homologous to each other, and that NLRR-1 protein is a transmembrane protein. Northern blot analysis and in situ hybridization histochemistry showed that both NLRR-1 and NLRR-2 mRNAs were expressed primarily in the central nervous system (CNS); NLRR-1 mRNA was also detected in the non-neuronal tissues such as cartilage, while NLRR-2 mRNA expression was confined to the CNS at all developmental stages. These results suggest that there is at least one LRR protein family in the mouse and that these molecules may play significant but distinct roles in neural development and in the adult nervous system.

Differential expressions of the eph family of receptor tyrosine kinase genes (sek, elk, eck) in the developing nervous system of the mouse

To examine the roles of the eph subfamily of receptor tyrosine kinase (RTK), we isolated mouse cDNAs for sek, elk, and eck and localized their mRNAs in the developing mouse, with particular reference to the CNS development, by in situ hybridization. sek mRNA is most abundantly expressed throughout development; sek was detected in the germinal layer of the embryonic CNS during mid- to late-gestation and was widely expressed in the early postnatal brain. elk was expressed in the mantle layer of the embryonic CNS and showed a distribution complementary to that of sek. Differential expression of sek and elk was also observed in the early postnatal cerebellum; sek was expressed in the Purkinje cells, while elk was detected in the granule cells. eck was moderately expressed in the germinal layer of the embryonic CNS at mid-gestation, but its expression decreased as development proceeded. These spatio-temporally different patterns of gene expression suggest that these RTKs have distinct roles in mouse development despite their structural homology.

Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system

We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages. This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system. Eos protein could interact with itself and Ikaros protein through its C‐terminal portion in the yeast two hybrid assay. These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.

Expression of sonic hedgehog signal transducers, patched and smoothened, in human basal cell carcinoma

In basal cell nevus syndrome (BCNS) patients, mutations of a gene, patched (ptc), which encodes a putative signal transducer of sonic hedgehog protein (SHH), were found and are thought to be one of the major causes of BCNS. The SHH signaling pathway is an important developmental pathway, and ptc protein (PTC) is a suppressive component serving as a receptor for the secreted SHH. Another transmembrane protein, smoothened (SMO), forms a complex with PTC and regulates this signaling pathway. Recent transgenic studies have strengthened the importance of the SHH signaling system in the etiology of basal cell carcinoma (BCC). In this study, we examined the expression patterns of mRNA for ptc and smo in two different BCC subtypes and normal skin. We found that the expressions of ptc and smo mRNA were enhanced in the tumor nests of the nodular BCC, especially at the advancing portions, but were under the detectable level in the superficial BCC cases examined, indicating that ptc and smo mRNA expressions might be associated with BCC tumor progression and divide the BCC histologic types into two subtypes, superficial and nodular types. In addition, no obvious signals for ptc and smo mRNA were detected in the normal human epidermis, appendages, or seborrheic keratosis, indicating that the abnormal proliferation of follicular epithelial cells caused by ptc, smo and/or other genetic changes, which also cause ptc and smo overexpressions, might result in BCC tumor formation.

Comparison of tissue distribution of two novel serine/threonine kinase genes containing the LIM motif (LIMK-1 and LIMK-2) in the developing rat

We previously isolated two novel serine/threonine kinase genes containing the LIM motif (LIMK-1 and LIMK-2) from a rat cDNA library. To examine the functions of these genes, we performed in situ hybridization in the developing rat nervous system. LIMK-1 and LIMK-2 mRNAs mostly co-localized during development and are expressed preferentially in the central nervous system during mid-to-late gestation but the signals decreased during the post-natal period. However, differential gene expression was observed in some nuclei in the CNS; LIMK-1 mRNA was intensely expressed in the facial motor nucleus, the hypoglossal nucleus, deep nuclei of the cerebellum and the layers 3, 5 and 6 of the adult cerebral cortex while only LIMK-2 mRNA was preferentially expressed in the some parts of the epithelium. In the nasal cavity, LIMK-1 and LIMK-2 mRNAs were expressed complementarily. Our results suggest that LIMK-1 and LIMK-2 may have different functions in these regions during development.

L3, a novel murine LIM-homeodomain transcription factor expressed in the ventral telencephalon and the mesenchyme surrounding the oral cavity

By reverse-transcription polymerase chain reaction method, we isolated a novel murine LIM-homeodomain gene, L3. In situ hybridization analyses revealed that L3 mRNA was localized to the ventral telencephalon and the mesenchyme surrounding the oral cavity of mouse embryo, suggesting that L3 may be involved in the region-specific differentiation of these areas.